Project A. Determine genetic susceptibility and immunopathological mechanisms contributing to idiopathic tooth root resorption, to include delineating the role of IRF8 mutations in increased susceptibility to multiple idiopathic root resorption. Background: We noted that BSP KO mice exhibit a tooth root resorption phenotype. Based on this finding, we sought to identify individuals exhibiting multiple idiopathic cervical root resorption, a familial pattern of periodontal disease with suggested genetic susceptibility. Following IRB approval from the University of Detroit Mercy School of Dentistry and NIH, dental/medical histories, x-rays, saliva samples, and extracted teeth were collected from a kindred (4 affected and 4 unaffected members) exhibiting multiple idiopathic cervical root resorption. On examination, the proband and the affected son and daughter exhibited severe root resorption of multiple teeth, with no other significant medical history. Micro-CT of exfoliated teeth revealed severe cervical root resorption distinct from tooth decay. Whole exome sequencing, using saliva from affected and unaffected family members, identified SNPs in ten candidate genes that co-segregated with the resorption phenotype, including a novel autosomal dominant missense mutation in Interferon Regulatory Factor 8 (IRF8). Primarily expressed in immune cells, IRF8 is a key regulator of inflammation and bone metabolism, and its repression mediates osteoclastogenesis by enhancing the activity of the transcription factor, nuclear factor of activated T cells c1 (NFATc1). The identified amino acid change (G388S) in IRF8 was localized to a highly conserved C-terminal motif, leading to altered serine phosphorylation motifs and phosphoserine binding domains, and is predicted to cause a large shift in 3D protein folding. These data suggest that the G388S mutation would impair IRF8 heterodimerization with other transcription factors including NFATc1, thereby producing overactive osteoclasts that target the periodontia. Consistent with these predictions, we noted that, compared to WT mice, Irf8-/- mice exhibited increased osteoclast activity in the periodontia, and accelerated femur and alveolar bone loss. Ongoing: In collaboration with Drs. Ozato and Holland, we are characterizing the periodontal phenotype in Irf8 KO mice and in patients with IRF8 mutations. Human IRF8WT and mutant constructs were overexpressed in Irf8 KO macrophages to map genome-wide IRF8 mutant binding sites and profile differentially regulated genes using ChIP/ChIPseq and RNAseq. Compared to hIRF8WT, the mutant hIRF8G388S isoform failed to inhibit NFATc1-dependent transcriptional activation of cathepsin-K reporter, suggesting that hIRF8G388S mutation promotes osteoclastogenesis. In agreement, hIRF8G388S isoform transduced into Irf8-/- bone marrow macrophages promoted increased osteoclast differentiation, resorption pits, and osteoclast-gene expression and dysregulated genes based on RNA-seq and CHIP-seq analyses. Manuscript in preparation for plans to submit in Sept. 2017. Project B. Disorders of mineralization: In collaboration with NIDCR clinical researchers and other IC clinicians, we have been examining individuals with mineralized tissue metabolism disorders for alterations in tissues/cells of the DOC complex. Mutations in key regulators of Pi/PPi. Mineralization of skeleton and teeth is tightly regulated by levels of extracellular inorganic phosphate (Pi) and pyrophosphate (PPi). Three regulators that control pericellular concentrations of Pi and PPi include tissue-nonspecific alkaline phosphatase (TNAP), progressive ankylosis protein (ANK), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). Inactivation of these factors results in mineralization disorders affecting teeth and their supporting structures. We examined the effect of decreased PPi on development and maturation of teeth in four human subjects with generalized arterial calcification of infancy (GACI), who harbor loss-of-function mutations in the ENPP1 gene. Three of the four subjects reported a history of infraocclusion or over-retained primary teeth or poor orthodontic tooth movement, suggesting altered mineral metabolism as a contributing factor. All subjects presented radiographic evidence of unusually protruding cervical root morphology in primary and/or secondary dentitions. Micro-CT analyses of extracted primary teeth from two GACI subjects revealed a marked increase in cervical cementum thickness and density vs. age-matched healthy control teeth. There were no differences in enamel and dentin densities between GACI and control teeth. Histology revealed dramatically expanded cervical cementum in GACI teeth, including cementocyte-like cells and unusual patterns of cementum resorption and repair. Micro-CT analysis of Enpp1 knock-out mouse molars revealed a marked increase in acellular cementum thickness and volume. Collectively, these findings report a novel dental phenotype in GACI and further support our hypothesis that Pi/PPi modulation is a key mechanism for regulating cementogenesis across species. Thumbigere-Math V, Alqadi A, Chalmers NI, Chavez MB, Chu EY, Collins MT, Ferreira CR, Fitzgerald K, Gahl WA, Ramnitz MS, Somerman MJ, Foster BL. Hypercementosis Associated with ENPP1 Mutations and Generalized Arterial Calcification of Infancy (GACI). JDR, 2017. In review. Ongoing: We are examining patients with Pi/PPi disorders at the NIH CRC and are obtaining teeth when extractions are required. In addition, we have obtained fibroblasts from GACI patients for in vitro evaluation of their mineralization capabilities. Project C. Chediak-Higashi Syndrome (CHS): Individuals with CHS, a rare autosomal recessive disease caused by mutations in the gene encoding lysosomal trafficking regulator, are immunodeficient, resulting in increased susceptibility to infections impacting several tissues, including oral tissues. In our ongoing collaboration with Drs. Nociti and Kantovitz from the State University of Campinas Brazil, seven CHS-diagnosed individuals were examined regarding their oral status in the NIH clinic, and gingival fibroblasts were obtained from atypical and classic CHS patients and control individuals. Clinical data indicated increased bleeding and probing depths of periodontal tissues vs periodontal tissues from healthy volunteers and more bleeding for atypical vs classic patients, yet the severe periodontal disease reported in the literature was not apparent. In vitro data reveal a different profile for atypical vs control cells at baseline and when challenged with E. coli LPS and F. nucleatum extract. Data from qPCR and multiplex protein analyses suggest that gingival fibroblasts from atypical patients exhibit an altered response to LPS suggestive of a more robust pro-inflammatory response to dental biofilm, and that higher levels of TLR-4 in atypical CHS GF play a key role in a hyperactive periodontal response to gram-negative bacteria (Thumbigere-Math et al., JDR CTR 2017).